Melanoma is arguably the most virulent among human cancers, in part due to its propensity to metastasize, and its resistance to conventional anti-cancer therapies. One key factor responsible for treatment failure relates to tumor heterogeneity, particularly subpopulations that possess stem cell-like properties, known as melanoma initiating cells (MICs). Our long-term goal is to understand the molecular mechanisms, whereby MICs and their stroma collaborate to promote growth and progression, as a means to develop effective therapeutic strategies. In the current project, we focus on defining the microenvironmental "niche", of CD133+/ABCB5+ MICs as a gateway to elucidate the complex cellular and molecular interplay that maintains the metabolic and replicative integrity of MICs. Our preliminary data indicate that: 1) the CD133+/ABCB5+ MICs are spatially arranged in a pattern identical to that of "vasculogenic mimicry (VM)";2) the vessel-like channels so formed in VM are themselves CD144 (VE-cadherin)+ and intimately associated with authentic endothelial structures, in keeping with so-called "perivascular niches";3) melanoma cell expression of bone morphogenetic protein 7 (BMP7) and its antagonist Noggin is associated with tumor progression and also co-localizes to areas of VM;and 4) BMP7 upregulates the angiogenic factor VEGF in the stromal microenvironment, but at the same time induces selective apoptosis in Noggin-deficient melanoma cells. Collectively, our findings support the central hypothesis that CD133+/ABCB5+ MIC-associated BMP7 may not only support the maintenance of MICs by facilitating "niche" morphogenesis (through coordinated VM and angiogenesis), but also balance metabolic demands (through stimulating VEGF-dependent angiogenesis and inducing selective apoptosis in the competitive, Noggin-deficient, non-initiating population). Using multi-label immunofluorescence and immunoguided laser capture microdissection followed by qRT-PCR, together with functional circulation analysis, we propose to further delineate the temporal/spatial relationship between melanoma cell CD133, ABCB5, BMP7 and CD144 expression, and the types/patterns of microcirculation (e.g. melanoma VM channel formation vs. angiogenesis) in vivo (Aim 1a). The dynamics and functional impacts of CD133+ melanoma subsets in "niche" formation (Aim 1b) will be tested in an orthotopic xenograft model using inducible RNAi-mediated knockdown at varying tempos. Finally, to dissect the CD133+/ABCB5+ MIC-associated BMP7 molecular signals in "niche" morphogenesis (Aim 2a) and tumor heterogeneity/homeostasis (Aim 2b), we will employ loss- and gain-of-function approaches to assess the BMP7-VEGF/BMP7-Noggin axes, in a tissue context in unique three-dimensional (3D) organotypic cultures in vitro and melanoma xenograft models in vivo. Defining mechanisms through which MIC-associated BMP7 signals contribute to "niche" development and maintenance offers a novel opportunity to therapeutically eliminate MICs directly or indirectly by targeting their stromal dependency. PUBLIC HEALTH RELEVANCE: Melanoma is notoriously resistant to conventional anti-cancer therapies, largely attributed to a subpopulation of cells with stem cell-like properties known as melanoma-initiating cells (MICs). The purpose of this study is to elucidate the mechanisms whereby MICs and their tissue environment collaborate to ensure survival and to promote tumor growth. We believe that defining such mechanisms offers a novel opportunity to therapeutically eliminate MICs directly or indirectly by targeting their dependency on tissue environment.